The long term objective of this research is to develop improved DNA polymerases that will substantially advance Sanger and "next generation" sequencing technologies. The primary goal of the Phase I research was to develop a rapid and inexpensive method to perform Sanger sequencing directly from single bacterial colonies. This method bypasses the time and expense of template purification needed for conventional DNA sequencing. It is based on the development of a modified Taq fusion DNA polymerase (DNAP) with improved DNA affinity and processivity. Chromosomal integration and expression of such a polymerase into host cells used for cloning should enable a new concept, termed "Ex cyto sequencing". Analogous to colony PCR, Ex cyto sequencing will eliminate the overnight growth of bacterial cultures, expensive template purification, and the purchase of purified DNA polymerase. In the Phase I research we developed a new fusion polymerase with novel enzymatic attributes, which was shown to improve multiple aspects of nucleic acid sequencing and amplification. In addition to sequencing trace amounts of DNA from a single bacterial colony, the new Taq fusion polymerase enabled the following procedures: sequencing difficult templates unresolved by other enzymes, sequencing directly from liquid cultures using as little as 5 ul of outgrowth media, tight DNA binding in a variety of buffer conditions, and long PCR (10 kb). The specific aims of the Phase II proposal are to complete the development and optimization of Ex cyto sequencing and to modify other DNA polymerases used in next generation sequencing, such as Bst, Klenow, and T4 DNAPs. These improved enzymes should provide superior read lengths and the ability to sequence through difficult structures, thereby improving the accuracy of base calling and the subsequent assembly of genomes. These enzymes will improve numerous nucleic acid synthesis applications, such as emulsion PCR, used for the in vitro clonal amplification of templates, whole genome amplification, long PCR amplification, and a host of other methods that are constrained by existing DNAP capabilities. PUBLIC HEALTH RELEVANCE: The success of the human genome project has spawned explosive growth in the demand for DNA sequence information. The discovery of new genes from a variety of species will have a large impact on understanding human health and disease. Despite improvements in speed and reduction in costs of DNA sequence analysis, the process is still time, labor, and cost-intensive. This proposal seeks to dramatically improve the speed while decreasing the costs of DNA sequencing by developing a next generation DNA polymerase.